Elevated WTAP promotes hyperinflammation by increasing m6A modification in inflammatory disease models

Emerging evidence has linked the dysregulation of N6-methyladenosine (m6A) modification to inflammation and inflammatory diseases, but the underlying mechanism still needs investigation. Here, we found that high levels of m6A modification in a variety of hyperinflammatory states are p65-dependent because Wilms tumor 1–associated protein (WTAP), a key component of the “writer” complex, is transcriptionally regulated by p65, and its overexpression can lead to increased levels of m6A modification. Mechanistically, upregulated WTAP is more prone to phase separation to facilitate the aggregation of the writer complex to nuclear speckles and the deposition of m6A marks on transcriptionally active inflammatory transcripts, thereby accelerating the proinflammatory response. Further, a myeloid deficiency in WTAP attenuates the severity of LPS-induced sepsis and DSS-induced IBD. Thus, the proinflammatory effect of WTAP is a general risk-increasing mechanism, and interrupting the assembly of the m6A writer complex to reduce the global m6A levels by targeting the phase separation of WTAP may be a potential and promising therapeutic strategy for alleviating hyperinflammation.

In vitro recombinant protein expression and purification.The expressing plasmids (pET-32a)   encoding WTAP conjugated eGFP and METTL3 conjugated mCherry were transformed into BL21 E.coli.E.coli were cultured in LB with Ampicillin (50 µg/mL) at 37 °C for about 12 hr till OD600 = 0.6.After induction with 1 mM IPTG at 37 °C for 8 hr, the cultured E.coli was harvested by centrifugation at 4000 rpm, 4 °C, 10 min and resuspended in lysis buffer.Cells were lysed by sonication on ice and centrifuged (12,000 rpm, 30 min, 4 °C) to remove debris and collected the supernatant.The supernatant was purified by incubation with Ni-NTA agarose beads (QIAGEN)   overnight at 4 °C.Then, Ni-NTA beads were washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 7.8), and proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 7.8).The purified proteins were further dialyzed by using PD10 column (GE Healthcare), and concentrated using Amicon Ultra 30 K (Millipore) concentrators at 4 °C.Concentrated proteins were quantified by the BCA method (Thermo Fisher) and stored at -80 °C.

ELISA assays and quantitative RT-PCR (qRT-PCR).
The concentrations of IL-6, IL-1β and TNF-α in culture supernatants were measured using kits from R&D Systems or Proteintech, according to the manufacturer's instructions.
Total RNA was extracted using TRIzol reagent (Invitrogen) and reversed-transcribed with a PrimeScript TM RT reagent kit with gDNA Eraser (TaKaRa) according to the manufacturer's instructions.And 2×Polarsignal ® qPCR mix (MIKX) was used for quantitative real-time PCR analysis.The data were normalized by the level of GAPDH or ACTB expression in each individual sample, and primer sequences used are listed in Supplementary Table 3.
Luciferase reporter gene assay.For a promoter reporter gene assay, 293T cells were plated in 48well plates and transiently transfected with WTAP promoter reporter (pGL3 basic plasmid), with increasing doses of Flag-tagged NF-κB p65, IRF3 or C/EBPβ and 7.5 ng of the Renilla luciferase reporter vector using Lipofectamine 2000.At 24 hrs post-transfection, luciferase activities were measured with a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions.Reporter gene activity was determined by normalization of Firefly luciferase activity to Renilla luciferase activity.
For other reporter gene assays, wild-type and WTAP Δ1-20 293T cells or wild-type WTAP Δ1-77 THP-1 cells were plated in 48-well plates and transiently transfected with the indicated reporter vectors (psiCHECK TM -2) expressing wild-type or mutated UTRs of IL6ST using Lipofectamine 2000.At 24 hrs post-transfection, the luciferase activities were measured as above.Reporter gene activity was determined by normalization of the Renilla luciferase activity to Firefly luciferase activity.
RNA decay assay.WTAP Δ1-77 and WT THP-1 cells were seeded at a density of 8 × 10 5 cells/mL in 12-well plates.After treatment with PMA (50 ng/mL, Sigma), the cells were treated with transcription inhibitor Act D (5 μg/mL, Sigma) to block de novo RNA synthesis, and collected at different times.RNA samples were extracted for qRT-PCR to determine the mRNA levels of the indicated genes.
Fluorescence recovery after photobleaching (FRAP) assay.FRAP assay was conducted by Leica TCS SP8 STED 3X confocal microscopy.488-or 568-nm laser beam was used to bleach the fluorescent protein at a region of interest (ROI), followed with collecting time-lapse images.
Fluorescence intensity of indicated ROI was measured and normalized to the fluorescence intensity of pre-bleaching image by Leica AS Lite.

Laser scanning confocal microscopy (LSCM).
Cells for fluorescence experiments were cultured in glass bottom culture dishes (Nest Scientific).After treated with indicated stimulation, cells were fixed by 4% paraformaldehyde for 10 min, followed with 3 times wash with PBS.Then, cells were permeabilized with methyl alcohol for 30 min at −20 °C and rinsed with PBS for 3 times.After blocking in 5% goat serum for 1 h at room temperature, cells were incubated with primary antibodies at 4 °C, overnight.1 × PBST (PBS with 0.1% Tween20) was used to wash the cells for 3 times and subsequently incubated with fluorescently labeled secondary antibodies at room temperature for 1 h.
Confocal images were obtained by Leica TCS SP8 STED 3X confocal microscope.The number of condensates were counted and analyzed by ImageJ software.
Live-cell imaging.At 24 hrs post-transfection incubation, cells were loaded into temperature-and CO2-controlled live-cell imaging chamber of Leica TCS SP8 STED 3X confocal microscope.Cells were imaged typically by use of 2 laser wavelengths (488 nm for eGFP imaging and 560 nm for mCherry imaging).
In vitro phase separation assay.The purified recombinant proteins were mixed at indicated concentration with LLPS buffer and 5% PEG8000 at 37 °C.The mixture was pipetted onto glass bottom dish and imaged by Leica TCS SP8 STED 3X confocal microscope equipped with m 6 A dot blots.Total RNA was extracted with TRIzol reagent.Equal amounts of RNA (300 ng) were denatured at 95 °C for 3 min.The samples were immediately chilled on ice, added to a positively charged nylon membrane (PALL), and then cross-linked with a UV crosslinker.After blocking and incubating with the m 6 A antibody (CST) overnight, the membrane was incubated with the secondary antibody at room temperature for 1 hr.Signals were detected using a chemiluminescence imaging system.Methylene blue in 0.3 M sodium acetate (pH 5.2) was used to indicate the amount of total RNA.

RNA immunoprecipitation assay (RIP). RIP was conducted using a RIP assay Kit (MBL)
following the manufacturer's instructions.In brief, Protein A/G magnetic beads coated with 5 μg of specific antibody or normal IgG were incubated with cell lysates at 4 °C overnight.Proteins were then extracted for immunoblot analysis, and the co-precipitated RNAs were isolated by elution buffer and purified by TRIzol reagent, and subsequently subjected to qRT-PCR analysis.
DNA/RNA pull-down assay.Biotin-labeled DNA and RNA probes were synthesized by RiboBio.
For DNA pull-down assay, Flag-tagged NF-κB p65 expression constructs were transfected into 293T cells.At 24 hrs post-transfection, whole cell lysates were extracted from 293T cells using IP lysis buffer.Biotin-coupled DNA-protein complex was pulled down by incubating whole cell lysates with high-capacity streptavidin agarose beads (Thermo Fisher) according to the manufacturer's instructions.The bound proteins were eluted and used for immunoblot analysis.
For RNA pull-down assay, biotin-coupled RNA complex was pulled down by incubating cell lysates with high-capacity streptavidin agarose beads, bound proteins were then extracted for immunoblot analysis.RNA-seq and data analysis.Whole-cell total RNA was isolated using TRIzol reagent and quantified using a NanoDrop 2000 spectrophotometer (Thermo).The cDNA library was constructed by Biomarker Technologies.Sequencing was performed on an Illumina HiSeq 2500 platform.Highquality reads were mapped to the human reference genome (hg19) or mouse reference genome (mm9) using HISAT2.DESeq, an R package, was applied for differential gene expression analysis.

Quantification of the m
We filtered the differentially expressed genes based on a false discovery rate (FDR) <0.05.

Characterizations of WTAP-deficient cells
To determine the function of WTAP in inflammatory responses, we first generated WTAP-knockout THP-1 cells using the CRISPR/Cas9 approach.Two sgRNAs were designed to target the second and third exons (E2 and E3) of WTAP.sgRNA#1 caused a two-base insertion, whereas sgRNA#2 caused a one-base deletion in the WTAP genomic locus (Supplementary Figure 3, A and B), leading to the introduction of a new stop codon upstream of the CDS and resulting in the early termination of WTAP translation.However, based on the predicted open reading frame (ORF), cells may skip these mutations by initiating translation at an alternative site to produce alternative WTAP isoforms of 376 and 319 aa, respectively (Supplementary Figure 3C).A subsequent immunoblotting assay using a monoclonal antibody (mAb) purchased from Abcam confirmed the extremely low expression of these two isoforms (Supplementary Figure 3D).Thus, the cell lines were designated "WTAP Δ1-20 THP-1 cells" and "WTAP Δ1-77 THP-1 cells".In addition to generating WTAP-deficient THP-1 cells, we also used the same sgRNAs to knock out WTAP in 293T cells and successfully obtained clones using sgRNA#1.Genomic sequencing confirmed that sgRNA#1 targeted the second exon and introduced a four-base deletion, also resulting in the early termination of WTAP translation.

Characterizations of WTAP conditional KO mice
We next generated the Wtap conditional KO (CKO) mice by crossing Wtap flox/flox mice with mice expressing Cre recombinase under the control of the lysozyme 2 promoter (LyzM-Cre) (Supplementary Figure 3H).Genomic sequencing confirmed that the third exon was fully removed after crossing, which also resulted in the early termination of WTAP translation (Supplementary Figure 3, I and J).Similarly, the ORF prediction revealed that gene-edited BMDMs may also produce alternative WTAP isoforms with 319 aa (Supplementary Figure 3J).However, none of the purchased antibodies targeting WTAP could identify this predicted isoform (Supplementary Figure 3, K and L).Although commercially available antibodies for detection are not sufficient, the expression of WTAP Δ1-77 should be extremely low in BMDMs if it is present, as we have previously observed in THP-1 cells.

Functional deficiency of truncated WTAP Δ1-20 and WTAP Δ1-77
WTAP possesses a nuclear localization signal (NLS) at its N-terminus (Supplementary Figure 3C), and mutation of this signal can affect the entry of the protein into the nucleus and its function (2).
Since the NLS is missing in both alternative isoforms, the entry of these isoforms into to the nucleus may be affected.Nuclear-cytoplasmic extraction assays confirmed that WTAP Δ1-20 and WTAP Δ1-77 were more concentrated in the cytoplasm (Supplementary Figure 3, M and N).Moreover, LC-MS/MS assays indicated that ectopic expression of full-length WTAP but not the two alternative isoforms in WTAP Δ1-20 293T cells could increase the global m 6 A modification level (Supplementary Figure 3O).Similarly, the m 6 A modification levels in WTAP Δ1-20 and WTAP Δ1-77 THP-1 cells were lower than those in wild-type cells (Supplementary Figure 3P), and the same trend was observed in BMDMs from LyzM-Cre + Wtap Δ1-77 mice (Supplementary Figure 3Q).Due to the extremely low expression and the severely impaired function of WTAP Δ1-20 and WTAP Δ1-77 , the status of WTAP Δ1- 20 and WTAP Δ1-77 cells should be very close to that of cells in which the protein is completely knocked out.Due to the extremely low expression and severely impaired function of WTAP Δ1-20 and WTAP Δ1-77 , the status of WTAP Δ1-20 and WTAP Δ1-77 cells should be very similar to that of cells in which the protein is completely deleted.
Additionally, no differences in the proportions of major immune cell populations were observed between Wtap fl/fl and LyzM-Cre + Wtap Δ1-77 mice in the steady state (Supplementary Figure 4, A-C  The predicted results were analyzed by the JASPAR (http://jaspar.genereg.net/).
6 A modification by LC-MS/MS.The 200ng extracted mRNA was digested into nucleosides by Nuclease P1 (1 U, NEB, M0660S) and shrimp alkaline phosphatase (rSAP, 1 U, NEB, M0371S) in 50 μL RNase-free water at 37 °C overnight.The mixture was diluted to 100 μL, 10 μL of which was injected into an LC-MS/MS system consisting of a high-performance liquid chromatographer (Shimadzu) equipped with a C18-T column (Weltech) and a Triple Quad 4500 (AB SCIEX) mass spectrometer in positive ion mode by multiple-reaction monitoring.Mass transitions of m/z 268.0-136.0(A), m/z 245.0-113.1 (U), m/z 244.0-112.1 (C), m/z 284.0-152.0(G) and m/z 282.0-150.1 (m 6 A) were monitored.A concentration series of pure commercial nucleosides (MCE) was employed to generate standard curves.The concentration of nucleosides in samples were obtained by fitting signal intensity to a standard curve with certain ratios calculated subsequently.
), indicating that the depletion of Wtap in myeloid cells did not affect macrophage development or maturation